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Abstract

Objective—Serum osteoprotegerin (OPG) concentrations have previously been associated with growth of abdominal aortic aneurysms (AAAs). In vitro experiments showed that OPG promotes matrix metalloprotease (MMP) release from monocytes and vascular smooth muscle cells. We hypothesized that OPG expression is increased in human AAAs and is associated with proteolysis.

Methods and Results—AAA biopsies were collected from 329 patients. We assessed the concentrations of OPG, cathepsins A, B, and S as well as the activity of MMP-2 and MMP-9. The AAA wall infiltration by macrophages, lymphocytes, and plasma cells was estimated by immunohistochemistry. The concentration of OPG correlated positively with aortic diameter (<55 mm: 16.1 [5.8–28.7], 55–70 mm: 21.9 [10.2–36.0], >70 mm: 24.0 [13.5–52.9] ng OPG/mg total amount of protein, P=0.020), cathepsin A (r=0.221, P=0.005), B (r=0.384, P<0.001), and S (r=0.467, P<0.001), MMP-2 (r=0.180, P<0.001), MMP-9 (r=0.178, P<0.001), and the number of lymphocytes (P<0.001) and plasma cells (P=0.001). OPG immunostaining was predominantly demonstrated in plasma cells.

Conclusion—The concentration of aortic wall OPG is positively associated with established markers of AAA severity and pathogenesis. OPG appeared to be associated with lymphocytes and plasma cells. These human data support previous experimental data suggesting a role for OPG in AAA pathogenesis.

Abdominal aortic aneurysm (AAA) is an abnormal focal dilation of the aortic wall that affects ≈5% of men aged >60 years. The risk of aortic rupture increases with AAA size, and AAA rupture is associated with a 80% to 90% mortality rate.1 Currently, the only treatment option for AAA is open or endovascular surgery; however, there is significant interest in developing medical therapies to limit AAA growth and rupture.2–4

Osteoprotegerin (OPG) is a secreted glycoprotein member of the tumor necrosis factor receptor superfamily.5 It acts as a decoy receptor for receptor activator of nuclear factor κB ligand (RANKL) and tumor necrosis factor–related apoptosis-inducing ligand.6 Cells described to secrete OPG include osteoblasts, endothelial cells, human aortic vascular smooth muscle cells (VSMCs), dendritic cells, lymphocytes, and plasma cells.7–9 OPG is a key regulator of bone remodeling5,10 but has also been implicated in tumorigenesis, immunologic pathways, and vascular diseases.6,7,11–15 The precise role of OPG in vascular disease is currently controversial. Evidence from animal studies suggests that OPG prevents arterial calcification and stabilizes plaque formation.16,17 However, clinical studies have associated serum OPG concentrations with the presence and progression of cardiovascular disease.18–21 Circulating OPG concentrations have also been reported to be higher in patients with AAA and positively associated with AAA progression.4,22 In vitro experiments have suggested that OPG stimulates matrix metalloprotease (MMP) secretion from human monocytes and VSMCs, and that OPG secretion is downregulated by irbesartan.4 These data suggest a potential role of OPG in AAA; however, currently we are aware of no studies that have reported the concentration of OPG in human AAA biopsies.

The aim of this study was to assess the concentration of OPG in a large number of AAA biopsies and assess whether OPG expression was related to markers of AAA severity. We hypothesized that OPG is locally produced in vessel walls of patients with AAA disease and is associated with markers of aortic proteolysis.

Methods

Aneurysm-Express Biobank

Aneurysm-Express is an ongoing longitudinal vascular biobank study.23 All patients undergoing open AAA repair in 2 Dutch hospitals are asked to participate in the study. The Medical Ethics Committees of both hospitals approved the study and participants provided written informed consent. For the current research investigation, we studied the ventral AAA wall biopsies from consecutive patients who were included between April 2003 and January 2011.

Patient Inclusion

In this study, consecutive patients undergoing open repair of intact or ruptured AAA were included. The indications for intervention were based on current guidelines and included the following: AAA diameter exceeding 55 mm for males, AAA diameters between 50 and 55 mm for females, rapidly expanding aortic diameters (≥5 mm in 6 months with a minimum diameter of 40 mm), saccular aneurysms, symptoms attributable to AAA and AAA rupture.1,24 Patients with AAA diameters between 50 and 55 mm were selected for surgery based on the clinical judgment of the surgeon and in consultation with the patient. Open repair was performed in patients in whom AAAs were not anatomically suited for endovascular repair. Patients with terminal malignancies or severe dementia were excluded from this study.

Baseline Characteristics

Risk factors and demographic data were obtained from clinical records and questionnaires at the time of recruitment. These questionnaires included history of vascular disease, cardiovascular risk factors (age, diabetes mellitus, sex, hypertension, smoking), and medication use. Aortic diameter and morphology were assessed via computed tomography angiography or magnetic resonance angiography. Hypertension was defined as systolic blood pressure >140 mm Hg or use of blood pressure–lowering drugs; diabetes mellitus was defined as the use of insulin or oral hypoglycemic agents; and smoking was defined on the basis of whether patients had smoked during the last weeks before AAA surgery. Chronic obstructive pulmonary disease was recorded based on previous physician diagnosis.25

Tissue Processing, Cell Culture, and Thrombus Volume Measurements

Comprehensive methods are available in the online-only Data Supplement.

Statistical Analysis

Continuous values were reported as median with interquartile range. Discrete variables were reported as number and percentages. Spearman’s correlation analysis and Kruskal-Wallis tests were conducted where appropriate. Multiple linear regression analysis was used to assess independent associations. To normalize the distributions and improve the linearity of the associations, the natural log transform was used during multiple linear regression analysis for non-normally distributed data. All tests were 2-sided, with P<0.05 considered statistically significant. Statistical analyses were performed with SPSS 15.0 (SPSS, Chicago, IL).

Results

Baseline Characteristics

AAA biopsies were collected from 329 patients at open surgery. The study cohort consisted of 258 asymptomatic, 35 symptomatic, and 36 ruptured AAAs. The baseline characteristics of the patients at the time of recruitment are shown in Table 1. There were 73 AAAs <55 mm, 162 AAAs between 55 and 70 mm, and 94 AAAs >70 mm. Patients with an aneurysm <55 mm were younger in age (P<0.001). No differences in comorbidities were observed.

A, Correlation of osteo protegerin (OPG) with abdominal aortic aneurysms (AAA) diameter in all AAA biopsies. B, Comparison of OPG concentrations in biopsies removed from AAAs with different diameters. OPG concentrations were higher in larger AAAs (*P=0.020). Data are expressed as mean±SEM.

OPG and Protease Expression in AAA

We measured extracellular matrix proteases because many serum proteases have been confirmed to be involved in the pathogenesis of experimental AAA models. MMP-2 and MMP-9 play an important role in AAA remodeling, and OPG stimulates MMP-2 and MMP-9 release from VSMCs and THP-1 cells in vitro.4 To investigate whether OPG correlates with MMP-2 and MMP-9 activities in AAA biopsies, we measured these markers using Biotrak assays. We measured cathepsins because of their known elastolytic and collagenolytic activity.26 Their active secretion can be regulated by VSMCs,27 and these cells are in direct contact with the elastic lamellae. High concentrations of cathepsins were detected in human AAA tissues, and these markers have been implicated to play a role in AAA pathogenesis.27–29 MMP-2 (r=0.180, P<0.001) and MMP-9 (r=0.178, P<0.001) activities were positively correlated with OPG concentration in the 329 AAA biopsies. There was also a significant correlation between the concentration of OPG in the 329 AAA biopsies and cysteine protease levels (Table 2). To our knowledge, these correlations were not reported earlier in AAA diseased tissues. OPG is the decoy receptor of RANKL, preventing nuclear factor κB activation. sRANKL stimulates MMP and cathepsin activation,7,30 which might have an effect on the integrity of the AAA wall. OPG might prevent high concentrations of sRANKL due to binding. However, sRANKL proved to be undetectable by Luminex technology. These data suggest that OPG is not a mere reflection of high sRANKL concentrations.

OPG and cathepsins showed a strong positive correlation in AAA biopsies. To investigate whether this correlation was indirect linkage or causally related, in vitro experiments were performed. Because of the observed correlations in our database among OPG, B lymphocytes, and cathepsin S, we stimulated B lymphocytes with rhOPG. Furthermore, VSMCs are in direct contact with the elastic lamellae and undergo apoptosis. VSMC apoptosis and elastic lamellae degeneration are considered important hallmarks of AAA disease. Therefore, we stimulated VSMCs with rhOPG. Healthy human abdominal aortic VSMCs showed a trend for increased upregulation of cathepsin S when incubated with increasing doses of rhOPG, P=0.076 (Figure 2A). A similar effect of rhOPG on secretion of cathepsin S from VSMCs was observed, P=0.074 (Figure 2B). No direct effects of rhOPG in vitro on secretion or upregulation of cathepsin S in B lymphocytes were detected (Figure I in the online-only Data Supplement). Simultaneous stimulation of B lymphocytes with rhOPG and IgM had no influence on the cathepsin S expression (data not shown). In addition, no direct effects of rhOPG in vitro on total B-lymphocyte proliferation were observed (Figure II in the online-only Data Supplement). Increasing doses of rhOPG stimulated cathepsin S upregulation in activated THP-1 cells, when cells were incubated in 0, 5, 10, and 20 rhOPG per 1×105 cells per 1 mL for 24 hours, respectively, P=0.049. A similar but nonsignificant effect of rhOPG on secretion of cathepsin S from activated THP-1 cells was demonstrated, P=0.092 (Figure III in the online-only Data Supplement).

OPG and Histology

Correlations of OPG with AAA diameter and markers of AAA severity were observed. OPG is expressed by numerous cells. Immunostaining for OPG was demonstrated to predominantly colocalize in the cytoplasm of plasma cells in AAA biopsies. Double immunohistochemical staining of OPG and CD138 in AAA biopsies confirmed this (Figure 3). Histological analyses showed that almost all immunologic cells were located in the adventitial layer, which is in line with previous reports.31 Therefore, we confined the correlations between measured OPG concentrations with macrophages, T lymphocytes, B lymphocytes, and plasma cells to the adventitial layer. The amount of these cells in the adventitial layer of the aorta in all histologically analyzed AAA biopsies is given in the online-only Data Supplement (Figures IV and V in the online-only Data Supplement). OPG correlated positively in AAA biopsies with staining of T lymphocytes (r=0.203, P=0.002), B lymphocytes (r=0.281, P<0.001), and plasma cells (r=0.329, P<0.001; Table 2). The AAA biopsy OPG concentrations were significantly different between staining categories for B and T lymphocytes, and for plasma cells (Figure 4). Also after correction for aneurysm diameter, age, sex, smoking, hypertension, history of myocardial infarction, chronic obstructive pulmonary disease, and diabetes mellitus, independent correlations remained. OPG concentrations were not associated with other histological characteristics of AAA biopsies (Table 3).

A, Immunohistochemical staining of osteoprotegerin (OPG) (shown in red) within the adventitia of the aortic wall. Bar=100 μm. B, Higher magnification of the indicated area in A. Bar=25 μm. C, Double immunohistochemical staining of OPG (in red) and CD138 (in blue). In the cells where CD138 is expressed on the cell membrane, OPG expression is present in the cell, confirming OPG expression in plasma cells. Bar=20 μm.

OPG and The Intraluminal Thrombus

OPG is present in the α-granules of platelets.34 Therefore, the intraluminal thrombus (ILT) could also be a source of secreted OPG. ILT volume and ventral thickness were measured. From 80 AAA patients preoperative computed tomography scans were directly available for ILT volume measurements. Analyses showed that OPG had no correlation with the ventral thickness of the thrombus (r=0.017, P=0.882) and ILT volume (r=0.163, P=0.148). However, in this subcohort, OPG had a borderline significant correlation with AAA diameter (r=0.218, P=0.052).

OPG Concentrations Among Asymptomatic, Symptomatic, and Ruptured AAAs

The heterogeneous nature of the AAAs might bias the results. Therefore, we performed a subanalysis of 258 asymptomatic, 35 symptomatic, and 36 ruptured AAA patients. Fewer ruptured AAA patients were diagnosed or treated for hypertension (Table 4). OPG concentrations among asymptomatic, symptomatic, or ruptured AAA patients did not differ (P=0.944). OPG was correlated with markers of AAA severity in asymptomatic AAA patients. Symptomatic and ruptured AAA patients did not show significant correlations with markers of AAA severity compared with the asymptomatic AAA patients; however, this might be caused by underpowering. An overview of scatter plots for OPG concentrations and AAA diameter concerning the asymptomatic, symptomatic, and ruptured AAA groups is given in the online-only Data Supplement (Table I and Figure VI in the online-only Data Supplement).

Discussion

The main finding from the present study was that aortic concentration of OPG was associated with a range of markers of AAA severity. OPG concentration was positively associated with AAA diameter, markers of proteolysis and inflammation in a large collection of human AAA samples. In an in vitro study we found that OPG appeared to stimulate the production of cathepsin S by VSMCs and monocytic cells, suggesting a possible reason for the link between OPG and cathepsin concentrations.

The correlation between AAA, OPG concentrations and aneurysm diameter remained significant after adjustment for traditional risk factors. These observations in human aortic biopsies are in line with previous observations in human blood samples. Moran et al4 previously found a correlation between serum OPG concentrations and AAA progression. Moran and colleagues also demonstrated that OPG stimulated MMP release from monocytes and VSMCs in vitro.4,35 In the present study, the aortic wall OPG concentrations and the activity of MMP-2 and MMP-9 were correlated in keeping with the previous findings of Moran et al. In the present study we also found correlations between the aortic concentrations of OPG and cysteine proteases, especially cathepsin S. To investigate this association further, we examined the effect of rhOPG on the expression of cathepsin S by VSMCs, B lymphocytes, and monocytic cells. These studies suggested that OPG promoted cathepsin S production in VSMCs and monocytic cells, although findings were of borderline significance. To our knowledge, the intracellular pathway via which OPG induces upregulation of proteolytic enzymes in these cells is unknown.

Zauli et al36 demonstrated increased adhesion and rolling properties of leukocytes in vitro and in vivo due to OPG administration. In the present study we found a significant correlation between concentrations of OPG and soluble intercellular adhesion molecular-1 in AAA biopsies. This finding is in keeping with a previous study from our group which demonstrated that OPG promoted expression of adhesion molecules by prestimulated endothelial cells.37 OPG was also associated with other markers of inflammation. Whether these correlations between OPG and pathological markers of AAA severity represent a causal link is not currently clear.

Immunodetection of OPG in AAA specimens localized the protein predominantly in the adventitia. OPG was demonstrated in plasma cells in the adventitial layer. AAA biopsy OPG concentrations were correlated with staining for B and T lymphocytes and plasma cells. There was no correlation of OPG with grading of VSMC numbers, elastin and collagen content. Most of the aortic media is degraded in advanced AAA disease, and therefore any link between elastin loss and OPG may be difficult to detect in the samples we analyzed. In keeping with our current findings, Li et al8 have previously reported that lymphocytes and plasma cells were the major sources of OPG in the bone microenvironment. Previous studies have implicated a role for OPG in regulating the humoral immune response, which has been suggested to play a role in AAA.11,38–40 OPG might promote an immune response within the aneurysm wall, via dysregulated cathepsin S/major histocompatibility complex II activation, and thereby influence B-cell activation, maturation, and isotype class switching.41–43 However, we could not show a direct effect of OPG on B-lymphocyte cathepsin S production in vitro. Therefore, it seems more likely that the correlation between OPG and cathepsin S is explained by common determinants, such as the presence of tertiary lymphoid organs in the adventitia.

OPG is a decoy receptor for sRANKL and therefore binds sRANKL in the extracellular matrix. OPG-sRANKL complexes cannot bind to cell receptors. Because sRANKL has effects on the immune and vascular system and can form a complex with OPG,6,14 we attempted to measure sRANKL expression in human AAA samples. sRANKL proved to be undetectable by Luminex technology. This could indicate low levels of sRANKL due to high levels of OPG or no production of sRANKL by the cells in the AAA vessel wall.

Several studies showed that OPG inhibits aortic calcification in mice.35,44–46 In the present study there was no significant correlation between aortic concentrations of OPG and calcification. A reason for this observation could be the stage of the AAA disease. Most of the media was degraded in the biopsies examined and calcification was primarily located in the intimal layer. To our knowledge, OPG has been associated predominantly with medial calcifications in mice.7,35 However, the data from our study suggest that OPG does not play an important role in calcification formation in advanced AAAs. Furthermore, whether OPG is causally related to AAA wall calcification is currently unknown. RANKL has been shown to affect VSMC physiology inducing osteogenic differentiation and calcification.7 OPG binds to RANKL, and thereby might inhibit (medial) calcification in human aortas or AAAs, but the mechanism has to be elucidated. However, Rhee et al47 failed to show an association of OPG polymorphisms with aortic calcification. On the contrary, RANKL polymorphisms showed an association with aortic calcification.48

Several limitations of our study need to be addressed. Whether OPG is causally related with advanced AAA disease remains unclear from this study. The results could suggest indirect linkage of OPG with proteolytic enzymes. The role of OPG-stimulated proteolytic enzymes in VSMCs and macrophages is probably limited in advanced AAAs, and may play a more central role in the early phase of AAA disease. In advanced AAA disease, OPG might have an influence on the adaptive immune system/tertiary lymphoid organs via B-lymphocyte isotype switching and maturation.11 Previous studies demonstrated a correlation between ILT volume and AAA volume increase. In addition, several studies investigated biological processes (oxidation, fibrinolysis, proteolysis) as a consequence of the thrombotic process and its effects on AAA progression.49 The AAA-express does not collect the ILT, and therefore we were not able to investigate the mechanisms to assess thrombus-related influences on the AAA vessel wall.

In conclusion, OPG is present in AAA biopsies and is predominantly expressed in adventitial lymphocytes and plasma cells. AAA wall OPG concentrations are associated with the concentrations of proteases and AAA diameter. The findings support previous work suggesting that OPG may be involved in AAA pathogenesis.

Acknowledgments

We thank Petra van der Kraak-Homoet for technical support.

Sources of Funding

Jonathan Golledge is supported by research funding from the National Health and Medical Research Council, the Queensland Government, and the Office of Medical Research, Australia. Dr Moran is supported by a Smart State Fellowship from the Queensland Government.

. The relationship between four single nucleotide polymorphisms in the promoter region of the osteoprotegerin gene and aortic calcification or coronary artery disease in Koreans. Clin Endocrinol (Oxf). 2006;64:689–697.